Title

Author

Date of Award

2005

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Neuroscience

First Committee Member

John N. Barrett, Committee Chair

Abstract

Hyperthermia can cause brain damage and exacerbate damage produced by traumatic brain injury, stroke, and amphetamines. The developing brain is especially sensitive to hyperthermia. However, little information is available on how heat damages neurons. I used cultured rat embryonic neurons to study hyperthermia-induced neuronal damage. This damage depended on the duration and temperature of the heat stress, as well as the age of the neuronal cultures. For a window of less severe heat stress (43°C for 2 hr), 1--2 week old cultured neurons produced no morphological signs of damage during the first 12 hr after the stress, but died over the next 3 days. Delayed activation of caspase-3 peaked in neurons by ∼18 hr and the general caspase inhibitors qVD-OPH and zVAD-fmk delayed neuronal death even when added with a 9 hr delay. In non-neuronal cells, hyperthermia can induce apoptosis via damage to the endoplasmic reticulum (ER), cytoskeleton, and/or mitochondria. I did not observe strong evidence for hyperthermia-induced ER, nuclear, or excitotoxic damage, but established mitochondrial dysfunction including two distinct changes in mitochondrial membrane potential (DeltaPsim) depolarization: a partially reversible depolarization during the heat stress and a later irreversible depolarization. Mitochondrial oxygen respiration was reduced by 50% by the end of the stress and did not recover. Results of experiments using the complex I inhibitor rotenone and the complex II and III substrates succinate and glycerol-3-phosphate suggest that the dysfunction that contributes to reduced respiration is likely upstream of complexes II/III. ATP levels were maintained, probably by glycolysis, within 25% of non-heated controls for 4.5 hr following the stress. Overexpression of the anti-apoptotic Bcl-2 family member, Bcl-xL, delayed neuronal death and slowed the disintegration of neuronal processes. The heat stress also induced cytochrome c release and activation of caspase-9. Finally, inhibitors of mixed lineage kinases and the MEK/ERK pathway enhanced neuronal survival following heat stress. My findings indicate a window of heat stress severity results in caspase-mediated apoptotic-like death, mitochondrial dysfunction, and likely kinase involvement. Uncovering these mechanisms of heat-induced damage may lead to therapeutic treatments to mitigate hyperthermia-induced or exacerbated damage and further the understanding of neuronal apoptosis.